E-Cigarette Aerosol Exposure Favors the Growth and Colonization of Oral Streptococcus mutans Compared to Commensal Streptococci

ABSTRACT E-cigarettes (e-cigs) have drastically increased in popularity during the last decade, especially among teenagers. While recent studies have started to explore the effect of e-cigs in the oral cavity, little is known about their effects on the oral microbiota and how they could affect oral health and potentially lead to disease, including periodontitis and head and neck cancers. To explore the impact of e-cigs on oral bacteria, we selected members of the genus Streptococcus, which are abundant in the oral cavity. We exposed the commensals Streptococcus sanguinis and Streptococcus gordonii and the opportunistic pathogen Streptococcus mutans, best known for causing dental caries, to e-liquids and e-cig aerosols with and without nicotine and with and without menthol flavoring and measured changes in growth patterns and biofilm formation. Our results demonstrate that e-cig aerosols hindered the growth of S. sanguinis and S. gordonii, while they did not affect the growth of S. mutans. We also show that e-cig aerosols significantly increased biofilm formation by S. mutans but did not affect the biofilm formation of the two commensals. We found that S. mutans exhibits higher hydrophobicity and coaggregation abilities along with higher attachment to OKF6 cells than S. sanguinis and S. gordonii. Therefore, our data suggest that e-cig aerosols have the potential to dysregulate oral bacterial homeostasis by suppressing the growth of commensals while enhancing the biofilm formation of the opportunistic pathogen S. mutans. This study highlights the importance of understanding the consequences of e-cig aerosol exposure on selected commensals and pathogenic species. Future studies modeling more complex communities will provide more insight into how e-cig aerosols and vaping affect the oral microbiota. IMPORTANCE Our study shows that e-cigarette aerosol exposure of selected bacteria known to be residents of the oral cavity hinders the growth of two streptococcal commensals while enhancing biofilm formation, hydrophobicity, and attachment for the pathogen S. mutans. These results indicate that e-cigarette vaping could open a niche for opportunistic bacteria such as S. mutans to colonize the oral cavity and affect oral health.

Introduction -The authors dedicate an entire paragraph to e-cig and periodontal disease. Still, the authors focus on S. mutans, which is associated with tooth decay and not with periodontal disease. This seems puzzling to me. Additionally, there does not seem to be any evidence of increased tooth decay due to the use of e-cigs. Thus, based on the introduction, I do not understand why the authors focus on S. mutans.
Additionally, It is unclear how focusing on three strep species grown as single species will help you better understand the oral microbiome. Maybe you should consider doing multi-species biofilms to see how e-cigs affect these streps when grown under slightly more complex conditions than just pure culture.

Methods -
Bacterial culture-Minor typo: it should be bacteria were sub-cultured in TSB, not "bacteria was..." Growth curve -I am particularly interested in the exposure of TSB to e-cig vapors. The authors mention that TSB was pretreated with e-cig aerosol (10-sec puff, 5-minute exposure). Was this done only once, or were this repeated multiple times? As written, it is unclear to me.
Biofilm formation -sanguinis and gordonii are lousy biofilm formers in the absence of a saliva-coated surface or sugar, like sucrose. To get a little closer to oral conditions, I would encourage the authors to coat their 96-well plates with saliva.

Results -
The authors are trying to make the case that a decrease in S. s and or S. g could favor the growth of S. m in the oral cavity. The authors should consider co-culturing the different steps and exposing the multi-species biofilm to e-cig vapor to determine any effect of the growth of the different species.
Page 12 -minor typo. I think the authors may want to say conducive instead of conductive.
Page 13 -The authors overstate the link between hydrophobic and host surface attachment, neglecting the fact that interactions with host surfaces can also be mediated by bacterial surface proteins. I encourage the authors to tone their conclusion down here because while hydrophobicity may contribute to surface attachment, it is not the only factor. In this study, Catala-Valentin et al. studied the impact of E-cigarette aerosol on the growth and biofilm formation of three oral streptococci. They showed that E-cigarette aerosol inhibited the growth of two commensal Streptococcus species, i.e., S. sanguinis and S. gordonii, but did not affect the growth of an oral pathogen, S. mutans. Most of the results shown in this study have been reported by previous publications. In the papers PMID: 31835369 and PMID: 33329035, the authors showed that the growth of oral commensal streptococci were inhibited by E-cigarette aerosol. In PMID: 32683796, E-cigarettes increased the growth and biofilm formation of S. mutans on teeth surfaces. More similar publications can be found. Hence, the authors need to show more innovative results.

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Introduction -The authors dedicate an entire paragraph to e-cig and periodontal disease. Still, the authors focus on S. mutans, which is associated with tooth decay and not with periodontal disease. This seems puzzling to me. Additionally, there does not seem to be any evidence of increased tooth decay due to the use of e-cigs. Thus, based on the introduction, I do not understand why the authors focus on S. mutans.
Response: On page 4 of the introduction, we included more rationale to why we focus on Streptococci and references to the literature highlighting the role of S. mutans not just in dental caries but also in periodontitis.
Additionally, it is unclear how focusing on three strep species grown as single species will help you better understand the oral microbiome. Maybe you should consider doing multi-species biofilms to see how e-cigs affect these streps when grown under slightly more complex conditions than just pure culture.
Response: While we added more references supporting our rationale to assess the effect of e-cig aerosols on the three Strep species we selected (page 4) as well as a new experiment to assess competition between S. mutans and the commensals ( Figure  S1). We show that S. mutans outcompetes S. sanguinis and gordonii (as has been shown by Kreth et al) and using 'conditioned media' from S. mutans culture that the suppression of commensal growth is due the secretion of metabolites by S.mutans into the media.
We agree with the reviewer that establishing multi-species model communities of higher complexity will be more valuable due to their physiological relevance, and we do plan to do so in future experiments.
Bacterial culture-Minor typo: it should be bacteria were sub-cultured in TSB, not "bacteria was..." Response: Thank you for catching that! Growth curve -I am particularly interested in the exposure of TSB to e-cig vapors. The authors mention that TSB was pretreated with e-cig aerosol (10-sec puff, 5-minute exposure). Was this done only once, or were this repeated multiple times? As written, it is unclear to me. Response: We clarified the preparation of TSB media exposed to e-cig aerosols in the Materials and Methods under E-cigarette aerosol exposure and the following paragraph "Growth Curve". Media were exposed once to a10-second puff for a 5-minute exposure before every experiment.
Biofilm formation -sanguinis and gordonii are lousy biofilm formers in the absence of a saliva-coated surface or sugar, like sucrose. To get a little closer to oral conditions, I would encourage the authors to coat their 96-well plates with saliva. Response: Thank you for your guidance on this! We performed additional biofilm experiments with 1% sucrose supplementation (new Figure S4). While the biofilm for both S. sanguinis and S. gordonii was enhanced compared to no-sucrose conditions, ecig aerosol exposure in the presence of sucrose did not enhance biofilm formation for the commensals.
The authors are trying to make the case that a decrease in S. s and or S. g could favor the growth of S. m in the oral cavity. The authors should consider co-culturing the different streps and exposing the multi-species biofilm to e-cig vapor to determine any effect of the growth of the different species. Response: Following your suggestion, we performed the competition experiments shown in new Fig S4. At this time, we can only show that S. mutans outcompetes S. sanguinis and gordonii (as has been shown by Kreth et al) and that the suppression of commensal growth is due to the secretion of metabolites by S. mutans into the media.
We have yet to grow multi-species biofilm and expose them to e-cig vapor. We agree with the reviewer this would be a more impactful experiment and we do plan to do so in future experiments.
Page 12 -minor typo. I think the authors may want to say conducive instead of conductive. Response: Thank you! Corrected! Page 13 -The authors overstate the link between hydrophobic and host surface attachment, neglecting the fact that interactions with host surfaces can also be mediated by bacterial surface proteins. I encourage the authors to tone their conclusion down here because while hydrophobicity may contribute to surface attachment, it is not the only factor. Response: We made changes to the text in the hydrophobicity section to highlight the value of the assay in its own without using it as the only read-out for cell attachment capabilities. In addition, we included more factors of changing hydrophobicity in the discussion. As can be appreciated from the images the clustered growth of S. mutans did not allow for proper quantification. We agree with the reviewer that in the absence of quantification this is not helpful but kept the images to demonstrate the difference in aggregation of S. mutans over the other two strains. We performed new attachment experiments to address the issue of quantification, plated cell lysate and bacteria upon co-culture and included conditions of e-cig exposure as suggested by the reviewer (new Figure 6). S. mutans had an overall increased capacity to attach compared to the commensals and appeared to be altered under some conditions of e-cig aerosol exposure but not significantly.
Reviewer #2 (Comments for the Author): In this study, Catala-Valentin et al. studied the impact of E-cigarette aerosol on the growth and biofilm formation of three oral streptococci. They showed that E-cigarette aerosol inhibited the growth of two commensal Streptococcus species, i.e., S. sanguinis and S. gordonii, but did not affect the growth of an oral pathogen, S. mutans. Most of the results shown in this study have been reported by previous publications. In the papers PMID: 31835369 and PMID: 33329035, the authors showed that the growth of oral commensal streptococci were inhibited by E-cigarette aerosol. In PMID: 32683796, E-cigarettes increased the growth and biofilm formation of S. mutans on teeth surfaces. More similar publications can be found. Hence, the authors need to show more innovative results.
Response: We appreciate the comments and feedback. We agree that the assessment of e-cig aerosol exposure of Strep. species is not novel. However, as we refer to the study findings listed by the reviewer throughout the manuscript, their analysis focused on one species of Strep. and could not be compared between each other. More so, as referred to some of these studies had conflicting results in terms of vape exposure modifying growth or with certain e-cig liquids (e.g., flavor, nicotine). Our study included all these conditions and the experiment for all species were done to be directly comparable.